Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients' malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.